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Biomedical subjects

I E Morrison

Publications and source records attributed to I E Morrison.

At least 19 recordsLinked to original sources
PubMed2000

Investigations of spectrin-lipid interactions using fluoresceinphosphatidylethanolamine as a membrane probe.

The binding of human erythrocyte spectrin to large unilamellar vesicles (LUVET) formed by the extrusion technique has been studied using fluoresceinphosphatidylethanolamine (FPE) as a reporter of electrostatic membrane potential. Spectrin aliquots were added to a suspension of FPE-labelled LUVETs to elucidate both the type of charge involved and the dissociation constants for spectrin binding to various lipids. All binding experiments showed serial increases in FPE fluorescence intensity upon serial additions of spectrin, indicative of increasing positive charge at the membrane surface. This proves for the first time that although exhibiting an overall net negative charge, spectrin binds to lipid surfaces by presenting positive charges to the lipid surface. Binding curves were obtained from the change in fluorescence intensity upon each spectrin addition and analysed to determine dissociation constants. A K(d) of 0.14+/-0.12 microM was found for spectrin binding to FPE-labelled phosphatidylcholine/phosphatidylserine (PC/PS) LUVETs at 22 degrees C in high salt conditions. A similar K(d) of 0.17+/-0.11 microM was obtained for spectrin binding to neutral LUVETs composed of PC. However, binding was found to be much weaker for PC/PS LUVETs under low salt conditions with a K(d) of 1.22+/-0.48 microM.

Buffers↗
PubMed2000

Digital fluorescence imaging of trafficking of endosomes containing low-density lipoprotein in brain astroglial cells.

We have used digital fluorescence microscopy to examine transport of LDL-containing endosomes in rat brain astroglial cells to show that individual middle endosomes undergo rapid transitions between forward/backward movements and immobile states over short distances. The population of rapidly moving endosomes (>0.04 microm/sec) was 35. 9%, and the remaining endosomes were slowly moving or temporarily immobile (<0.04 microm/sec). The averaged motion was, however, a very slow perinuclear motion with a velocity of 3.25 microm/h. This small velocity is mainly due to frequent changing of directions in movements, requiring 6 h for a significant concentration around the circumference of the cell nuclei. The application of both anti-dynein antibodies and vanadate in permeabilized cells resulted in peripherally concentrated distribution of endosomes, probably due to inhibition of perinuclear motion by dynein-like motor proteins. These results imply that both dynein-like and kinesin-like proteins bind to the same endosome resulting in both perinuclear and peripherally directed movements.

Animals↗
PubMed1999

Anomalous diffusion of major histocompatibility complex class I molecules on HeLa cells determined by single particle tracking.

Single-particle tracking (SPT) was used to determine the mobility characteristics of MHC (major histocompatibility complex) class I molecules at the surface of HeLa cells at 22 degrees C and on different time scales. MHC class I was labeled using the Fab fragment of a monoclonal antibody (W6/32), covalently bound to either R-phycoerythrin or fluorescent microspheres, and the particles were tracked using high-sensitivity fluorescence imaging. Analysis of the data for a fixed time interval suggests a reasonable fit to a random diffusion model. The best fit values of the diffusion coefficient D decreased markedly, however, with increasing time interval, demonstrating the existence of anomalous diffusion. Further analysis of the data shows that the diffusion is anomalous over the complete time range investigated, 4-300 s. Fitting the results obtained with the R-phycoerythrin probe to D = D0talpha-1, where Do is a constant and t is the time, gave D0 = (6.7 +/- 4.5) x 10(-11) cm2 s-1 and alpha = 0.49 +/- 0.16. Experiments with fluorescent microspheres were less reproducible and gave slower anomalous diffusion. The R-phycoerythrin probe is considered more reliable for fluorescent SPT because it is small (11 x 8 nm) and monovalent. The type of motion exhibited by the class I molecules will greatly affect their ability to migrate in the plane of the membrane. Anomalous diffusion, in particular, greatly reduces the distance a class I molecule can travel on the time scale of minutes. The present data are discussed in relation to the possible role of diffusion and clustering in T-cell activation.

Adenosine Triphosphate↗
PubMed1998

Mobility of cell surface receptors: a re-evaluation.

It has long been known from fluorescence recovery after photobleaching experiments that the mobility of most cell surface receptors is much smaller than expected for free diffusion of proteins in a fluid lipid bilayer. Single-particle tracking experiments are currently revealing the complexity of the constraints to free diffusion. Evidence has been obtained for several different processes: domain-limited diffusion, temporary confinement and anomalous diffusion. The type of motion exhibited by a given receptor will profoundly influence the rate of any functional process which requires movement in the plane of the membrane. In particular, anomalous diffusion greatly reduces the distance travelled by a receptor on a time scale of minutes.

Diffusion↗
PubMed1998

Detection of dimers of dimers of human leukocyte antigen (HLA)-DR on the surface of living cells by single-particle fluorescence imaging.

The technique of single-particle fluorescence imaging was used to investigate the oligomeric state of MHC class II molecules on the surface of living cells. Cells transfected with human leukocyte antigen (HLA)-DR A and B genes were labeled at saturation with a univalent probe consisting of Fab coupled to R-phycoerythrin. Analysis of the intensities of fluorescent spots on the cell surface revealed the presence of single and double particles consistent with the simultaneous presence of HLA-DR heterodimers and dimers of dimers. The proportion of double particles was lower at 37 degrees C than at 22 degrees C, suggesting that the heterodimers and dimers of dimers exist in a temperature-dependent equilibrium. These results are discussed in the context of a possible role for HLA-DR dimers of dimers in T cell receptor-MHC interactions. The technique is validated by demonstrating that fluorescence imaging can distinguish between dimers and tetramers of human erythrocyte spectrin deposited from solution onto a solid substrate. The methodology will have broad applicability to investigation of the oligomeric state of immunological and other membrane-bound receptors in living cells.

Cell Line↗
PubMed1997

Restriction by ankyrin of band 3 rotational mobility in human erythrocyte membranes and reconstituted lipid vesicles.

Rotational diffusion of eosin-5-maleimide-labeled band 3 was measured in erythrocyte membranes at pH 9.4-10.4. Band 3 was found to be more mobile in this pH range than at pH 7.5. Similar results were obtained with spectrin-actin-depleted membranes, where it was further shown that ankyrin is the only detectable protein released from the membrane at pH 10. Further experiments were performed at pH 7.5 to investigate the effects of rebinding purified ankyrin and/or band 4.1 to ghosts stripped of skeletal proteins. Ankyrin was found to reduce band 3 rotational mobility, but band 4.1 had no effect. A fluorescence binding assay revealed that fluorescein isothiocyanate-labeled ankyrin had similar binding parameters to those reported previously using 125I labeling. Finally, the rotational mobility of purified band 3 reconstituted into lipid bilayers was determined before and after ankyrin binding. The results of these reconstitution experiments were globally analyzed, assuming the existence of two populations of band 3 with different correlation times. The faster correlation time is consistent with that expected for either dimers or compact tetramers of band 3. Ankyrin binding reduces the proportion of band 3 contributing to the faster component. This result demonstrates that ankyrin promotes the association of band 3 into more slowly rotating complexes independently of any other components of the erythrocyte membrane. It has been reported that ankyrin contains two binding sites for band 3 [Michaely, P., & Bennett, V. (1995) J. Biol. Chem. 270, 22050-22057]. The results of the present study are thus explained by the ability of ankyrin to cross-link band 3 into larger diameter complexes. Cross-linking by ankyrin in part accounts for the slow components in the anisotropy decays of band 3 in the erythrocyte membrane. Other factors which probably influence band 3 aggregation include the membrane "fluidity" and protein concentration.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1996

Single particle tracking of cell-surface HLA-DR molecules using R-phycoerythrin labeled monoclonal antibodies and fluorescence digital imaging.

The mobility of cell surface MHC molecules and their ability to form dynamic associations may be related to the physiological status of the cell and to the potential to bind effector T lymphocytes. To investigate these properties, we have prepared HLA DR specific monoclonal antibodies coupled in a 1:1 mole ratio to the fluorescent phycobiliprotein, R-phycoerythrin (PE). We show that these small particles can be sequentially imaged using a cooled slow-scan charge coupled device camera and hence can be used for single particle tracking experiments. We have applied this technique to investigate the movements of HLA DR molecules on fibroblasts transfected with human DR alpha and DR beta genes. PE-IgG was bound to the transfected fibroblasts and particle tracks were obtained by sequential imaging over a period of typically 30 minutes. Analysis of particle tracks revealed the presence of directed motion and domain-limited diffusion in addition to random diffusion. The contributions of these three types of motion showed cell to cell variability. Velocities of directed motion were of the order of 2 nm second-1 whilst domain diameters were in the range 200-800 nm. Diffusion coefficients for random diffusion were in the range 1 x 10(-13)-5 x 10(-12) cm2 second-1. The higher mobilities were observed for the lower intensity fluorescent spots, which possibly correspond to images of single particles. Much lower mobility was observed with a cell where the spot intensities were approximately double that of the lower intensity spots. These spots could be images of double particles implying the association of at least two HLA DR alpha beta dimers. These data are relevant to the study of MHC class II cell surface redistribution and antigen presentation in specific immunity.

Antibodies, Monoclonal↗
PubMed1994

Analysis of receptor clustering on cell surfaces by imaging fluorescent particles.

Fluorescently labeled low density lipoproteins (LDL) and influenza virus particles were bound to the surface of human fibroblasts and imaged with a cooled slow-scan CCD camera attached to a fluorescence microscope. Particles were also imaged after attachment to polylysine-coated microscope slides. The digital images were analyzed by fitting data points in the region of fluorescent spots by a two-dimensional Gaussian function, thus obtaining a measure of spot intensity with correction for local background. The intensity distributions for particles bound to polylysine slides were mainly accounted for by particle size distributions as determined by electron microscopy. In the case of LDL, the intensity distributions for particles bound to fibroblasts were considerably broadened, indicative of clustering. The on-cell intensity distributions were deconvolved into 1-particle, 2-particle, 3-particle, etc. components using the data obtained with LDL bound to polylysine-coated slides as an empirical measure of the single particle intensity distribution. This procedure yielded a reasonably accurate measure of the proportion of single particles, but large errors were encountered in the proportions of larger cluster sizes. The possibility of studying the dynamics of clustering was investigated by binding LDL to cells at 4 degrees C and observing changes in the intensity distribution with time after warming to 20 degrees C.

Biophysical Phenomena↗
PubMed1992

Tracking of cell surface receptors by fluorescence digital imaging microscopy using a charge-coupled device camera. Low-density lipoprotein and influenza virus receptor mobility at 4 degrees C.

A fluorescence imaging system, based on using a cooled slow-scan CCD camera, has been developed for tracking receptors on the surfaces of living cells. The technique is applicable to receptors for particles such as lipoproteins and viruses that can be labeled with a few tens of fluorophores. The positions of single particles in each image are determined to within 25 nm by fitting the fluorescence distribution to a two-dimensional Gaussian function. This procedure also provides an accurate measure of intensity, which is used as a tag for automated tracking of particles from frame to frame. The method is applied to an investigation of the mobility of receptors for LDL and influenza virus particles on human dermal fibroblasts at 4 degrees C. In contrast to previous studies by FRAP (fluorescence recovery after photo-bleaching), it is found that receptors have a low but measurable mobility at 4 degrees C. Analysis of individual particle tracks indicates that whilst some receptors undergo random diffusion, others undergo directed motion (flow) or diffusion restricted to a domain. A procedure is proposed for subdividing receptors according to their different types of motion and hence determining their motional parameters. The finding that receptors are not completely immobilised at 4 degrees C is significant for studies of receptor distributions performed at this temperature.

Biological Transport↗
PubMed1990

Rotational diffusion studies of the lipoyl domain of 2-oxoacid dehydrogenase multienzyme complexes.

Rotational mobility of the lipoyl domain of a number of 2-oxoacid dehydrogenase complexes was investigated by transient dichroism after the domain had been specifically labeled with the triplet probe eosin-5-maleimide. Complexes investigated included pyruvate dehydrogenase complexes from Bacillus stearothermophilus, ox heart, and Escherichia coli (in which the E2 component had been genetically engineered to contain one lipoyl domain) and 2-oxoglutarate dehydrogenase complexes from ox heart and E. coli. Measurements were also performed with ox heart pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes specifically labeled on E1. Anisotropy decays were recorded in glycerol-buffer solutions of varying viscosity and at different temperatures. For E2-labeled complexes, the decays were found to be multiexponential, and the fastest correlation time was considerably shorter than expected for tumbling of the whole complex. This fast correlation time was absent from E1-labeled complexes and was assigned to independent motion of the lipoyl domain. Plots of the fast correlation time against eta/T showed a surprisingly weak dependence on viscosity and extrapolated to a time of 30-40 microseconds at zero viscosity. To explain this result, a model is proposed in which the lipoyl domain is in equilibrium between "free" and bound states. The time of 30-40 microseconds is shown to correspond to 1/koff, where koff is the rate constant for dissociation of the domain from binding sites on the complex. This dissociation phenomenon only contributes to the anisotropy decay when the viscosity of the solution is sufficiently high to slow the tumbling of the whole complex to times that are long in comparison to 1/koff.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1990

Transient dichroism studies of spectrin rotational diffusion in solution and bound to erythrocyte membranes.

Spectrin was purified from human erythrocytes and labeled with the triplet probe eosin-5-maleimide. Rotational diffusion of spectrin was investigated by observing transient dichroism following flash excitation of the probe. Measurements were performed at 4 degrees C in solutions of varying viscosity and with spectrin rebound to spectrin/actin-depleted erythrocyte membranes. In solution, complex anisotropy decays were observed which could not be satisfactorily fitted by the equations for a rod-shaped molecule of appropriate dimensions. When spectrin was rebound to the erythrocyte membrane, a decay in the anisotropy was still present but was markedly less sensitive to solution viscosity and flatter at longer times. In order to overcome the objection that the cytoskeleton is only partially reconstituted when spectrin is rebound, a method was developed for labeling spectrin with eosin-5-maleimide in situ. Anisotropy decays for these labeled membranes exhibited features similar to those obtained for spectrin labeled in solution and subsequently rebound. Taken together, the results provide good evidence for segmental motion of spectrin when incorporated into the erythrocyte cytoskeleton. Upon increasing the temperature, the initial anisotropy ro for both rebound and in situ labeled spectrin decreases, and above 30 degrees C the measured anisotropies are small. Thus, at physiological temperature the probe is almost completely randomized by motions with correlation times less than 10 microseconds.

Circular Dichroism↗
PubMed1989

Digital fluorescence imaging of fusion of influenza virus with erythrocytes.

Fusion of influenza virus with human erythrocytes at pH 5.2 was followed by fluorescence microscopy using a cooled slow-scan CCD camera. The high sensitivity of the CCD permits repetitive digital imaging of the same cells with minimal photobleaching. The experimental conditions were such that only a small number of virus particles were adsorbed per cell. Quantitative analysis of the data indicated that for most cells only a single fusion event took place. This was, however, sufficient to cause haemolysis within 30 min at 20-22 degrees C for about 60% of cells. There was a highly variable time lag between fusion and haemolysis. The lateral diffusion coefficient of virus particles on the cell surface when bound at pH 7.4 was less than 2 x 10(-13) cm2.s-1. The technique should be of value for more detailed studies of the dynamics of viral and other membrane fusion events.

Erythrocytes↗